Cytokinesis, the physical division of one cell into two daughter cells, is the final stage of the cell reproductive cycle and the least well understood. Correctly timing the process of cytokinesis so that it occurs only after chromosome replication and segregation is necessary to prevent catastrophic genomic instability and accordingly, cytokinesis is strictly regulated in concert with other events of the cell cycle. We have made significant progress in identifying and characterizing proteins essential for cytokinesis using a powerful model organism for cytokinesis studies, the fission yeast Schizosaccharomyces pombe. We now propose to gain a better understanding of how these myriad proteins work together and under cell cycle control to mediate successful cell division. We focus on two essential, conserved proteins that are necessary for assembling an actomyosin-based contractile ring that is used to pinch cells in two - 1) Cdc15 and 2) Cdc12. Cdc15 is a scaffold of the contractile apparatus and it links the actomyosin contractile ring to the plasma membrane through its F-BAR domain. We found that bulk dephosphorylation of Cdc15 at mitotic entry induces a conformational switch in the protein that allows it to oligomerize, bind the membrane and act as a stable membrane-anchored scaffold for cytokinetic ring assembly. We will now determine how Cdc15 oliogomerizes on membranes, and how other contractile ring components are organized around this scaffold using sophisticated microscopy approaches. Cdc12 is the formin that nucleates the F-actin of the contractile ring but how it is regulated is unclear. We defined new functional domains and phosphoregulation within Cdc12 and here we will test whether these additional domains and phosphorylation events modulate the ability of Cdc12 FH1-FH2 to nucleate, possessively elongate, and bundle F-actin using spontaneous assembly and sedimentation assays, and multi-color single molecule TIRF imaging with dual labeling of actin and Cdc12 fragments. We will also test our hypothesis that polarity kinases inhibit the establishment of the Cdc15 scaffold at cell ends, forcing it to assemble in the cell middle. We will complement these focused mechanistic studies with proteomic and large-scale genetic screens designed to establish a functional interaction network of cytokinesis components. Although some of the details will vary between organisms, these studies will have a major impact for understanding how cytokinesis is orchestrated in multiple species including humans.